Agriculture and Food Systems - Theses

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    Impact of Silicon on tolerance mechanisms of wheat (Triticum aestivum L.) under drought and heat stress environments
    Ashfaq, Waseem ( 2022)
    Bread wheat (Triticum aestivum L.) is widely cultivated and amongst the major staple food crops of the world. With a total grain production of around 760 million tonnes in 2020, the crop provides ~ 20 percent of the total dietary calories and protein to the human population around the world. However, global wheat productivity faces challenges due to climatic adversities, which are becoming more acute in most of the world's established agricultural regions, raising concerns for future food security. Abiotic stress factors such as drought and heat are the primary causes of crop grain yield reduction worldwide, and the frequency of their concomitant effects has increased in the semi-arid wheat belts of the world. Plant available silicon (Si) has been widely reported for its beneficial effects on plant development, productivity and attenuating physiological and biochemical impairments caused by various abiotic stresses. This research work investigated the impact of Si application on the morphological, physiological, and biochemical mechanisms in contrasting bread wheat cultivars at critical growth stages under individual and combined drought and heat stress. Firstly, a preliminary screening experiment was conducted to categorize 46 wheat genotypes, mostly from Australia, for terminal drought and heat stress tolerance. The stress tolerance level of each genotype was determined on the basis of morphological and physiological traits, including rapid, non-destructive infrared thermal imaging for computational water stress indices (Tc, CTD, Ig, Tdry, Twet, and CWSI). Multivariate data analysis on significant traits was performed to group drought and heat stress-tolerant and susceptible genotypes. Based on their overall performance, this study identified the top ten best and five lowest-performing genotypes for drought and heat stress tolerance. After identifying the drought and heat tolerance level of wheat cultivars, a series of experiments were conducted to evaluate the role of Si on drought and heat stress susceptible and tolerant wheat cultivars at critical growth stages. In this series, to evaluate the role of Si on the wheat root system and canopy physiology under drought stress, an experiment was conducted with two contrasting bread wheat cultivars (RAC875, drought tolerant; Kukri, drought susceptible) in the glasshouse. Results showed that compared with control (drought and no Si), Kukri had a significant increase in primary root length (PRL, 44 percent) and lateral root length (LRL, 28.1 percent) with Si treatment under drought stress compared with RAC875 having a significant increase in PRL (35.2 percent), but a non-significant increase in LRL. An increase in the wheat root system positively impacted the canopy physiology (photosynthesis, stomatal conductance, and transpiration) and computational water stress indices (Tc, CWSI, CTD, Ig) in Kukri and RAC875 under drought stress. These results showed that Si has the potential to influence below-ground traits, which regulate the moisture uptake ability of roots for a cooler canopy in tolerant and susceptible wheat cultivars under drought stress. Subsequently, a comparative study of drought tolerant (RAC875) and drought susceptible (Kukri) wheat cultivars investigated the impacts of pre-sowing Si treatment in attenuating the physiological and biochemical disruptions caused by pre-anthesis drought stress. The results showed, compared to the controls (drought and no Si), Si application significantly improved the relative water content (RAC875, 10.8 percent; Kukri, 18.1 percent), chlorophyll content (RAC875, 8.7 percent; Kukri, 12.7 percent), and chlorophyll fluorescence (RAC875, 10.1 percent; Kukri, 22.3 percent) in Si treated plants under drought stress. Similarly, the concentrations of various osmolytes and antioxidants increased with Si treatment in drought tolerant and susceptible cultivars under drought stress. Results showed that the impact of a Si-induced percent increase in physiological and biochemical traits was higher in Kukri (susceptible wheat cultivar) than tolerant wheat cultivar (RAC875) under pre-anthesis drought stress. Overall, these results showed that Si has the potential to enhance plant morphological, physiological, and biochemical traits and alleviate oxidative damage by improving antioxidant defense mechanisms, both in tolerant and susceptible wheat cultivars, during pre-anthesis drought stress conditions. Following this, another experiment was conducted to study the impact of pre-sowing Si treatment on the individual and the cumulative effects of drought and heat stress during later growth stages of contrasting wheat cultivars (drought and heat stress-tolerant, RAC875, Excalibur, ECH957, RAC622; drought and heat stress-susceptible, Kukri, CM59443). Results showed that Si treatment significantly improved various stress-affected morphological, physiological, and biochemical traits, including grain yield (tolerant wheat cultivars, > 40 percent; susceptible wheat cultivars, > 31 percent) and yield components. The highest averaged Si-induced increase in thousand-grain weight across all the stress treatments was observed in Excalibur (8.6 percent), followed by Kukri (6.9 percent) and CM59443 (4.9 percent). With Si treatment, osmolytes concentrations increased significantly by > 50 percent in tolerant and susceptible wheat cultivars. Similarly, computational water stress indices also improved with Si treatment under drought, heat and drought-heat combined stress in susceptible and tolerant wheat cultivars. The study concludes that Si treatment has the potential to mitigate the detrimental effects of individual and combined stress of drought, heat, and drought-heat combined stress at early grain-filling stages in susceptible and tolerant wheat cultivars in a controlled environment. Finally, to fully understand the role of Si under natural drought and heat-stressed field conditions, a field experiment was conducted at International Maize and Wheat Improvement Center (CIMMYT), Mexico. The study aimed to determine the effects of Si application on the performance of contrasting wheat cultivars (five checks, eight tolerant, and three susceptible to drought stress) in response to terminal drought and heat stress under field conditions. Results showed a significant (p < 0.05) Si effect on most of the measured agronomic and physiological traits, including improved grain yield (p < 0.01) under drought and heat-stressed environments. Relative grain yield gain with Si application among susceptible cultivars ranged from 7.8 percent to 61.4 percent, compared with 4 to 44 percent among the tolerant cultivars under drought and heat-stressed conditions. A significantly (p < 0.05) lower mean canopy temperature was observed in Si-treated plots compared with the control for both tolerant and susceptible cultivars. The stay-green phenotype at the mid-grain-filling stage, which was estimated using relative NDVI decay and relative rate of flag leaf greenness decay (based on SPAD measurements), was significantly (p < 0.01) enhanced in tolerant (9.7 percent) and susceptible cultivars (6.3 percent). Based on these findings, it can be concluded that Si application under field conditions significantly improved various agronomical and physiological traits both under drought and heat stress. Overall, the findings from the thesis revealed that Si has the potential to mitigate critical growth stage individual and combined effects of drought and heat stress in wheat cultivars through improved morphological, physiological, and biochemical attributes. The findings also revealed that Si application has the potential to enhance the tolerance level of susceptible wheat cultivars under critical growth stage drought and heat stress. This study suggests that Si addition as a nutritional element can potentially be a sustainable management strategy to mitigate individual and combined effects of drought and heat stress on susceptible and tolerant wheat cultivars in the rainfed cropping system.
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    Investigation of the interaction of sugarcane phenolics and fiber and the effects on gut microbiota
    Loo, Yit Tao ( 2022)
    During the last two decades, there has been an increasing interest in the role of gut microbiota in human health. The gut microbiota is the dynamic and complex microbial community that resides along the human gastrointestinal tract. Diet is one of the crucial factors in the establishment and regulation of the gut microbiota because various dietary compounds can interact with it in a different way and can cause significant impacts on the microbial activity and composition. Polyphenols and fiber that are commonly found in plant-based diets may contribute to the beneficial effects of the diets on gut microbiota enterotypes associated with good health. Sugarcane bagasse, a waste product from the processing of sugarcane, is known to be rich in fiber that could be beneficial to human health through the production of short chain fatty acids from bacteria fermentation. Sugarcane polyphenols have been shown to inhibit digestion of sucrose and starch and to inhibit absorption of glucose from the gut, modulating postprandial hyperglycaemia. Moreover, these polyphenols may have the ability to selectively stimulate the growth and activity of beneficial gut bacteria and simultaneously inhibit pathogenic bacteria likewise. Therefore, there is a possibility for sugarcane fiber and polyphenols to act in concert or synergistically in mitigating gut dysbiosis and altering the gut microbiota. One of the major difficulties in presenting extracted or purified polyphenols to the colonic environment from oral route is to escape modification, degradation and absorption in the stomach and small intestine. Using carriers such as dietary fiber might overcome such restrictions. Thus, the aim of my PhD research is to study whether sugarcane fiber (SCFiber) can behave as a carrier of sugarcane polyphenols to the colon and whether they can then selectively modify pig gut microbiota studied in an in vitro system. I begin by investigating the bio-accessibility of phenolic compounds to the colon after in vitro sequential gastric-intestinal digestion, using SCFiber in combination with a sugarcane crude extract (Phytolin) and a polyphenol rich sugarcane fraction (Polynol). The combinations of SCFiber with either Phytolin or Polynol showed a substantial increase in phenolic materials available for colonic fermentation after the digestions. I then move on to examine the effects of Phytolin, Polynol, SCFiber and their respective combinations on the gut microbiota profile and on short-chain fatty acid (SCFA) production in an in vitro pig fecal fermentation system. These samples are found to have modulatory impacts on the gut bacterial community to different extents, including alterations on the alpha-diversity and beta-diversity. Furthermore, they regulated the gut microbiota composition by affecting the relative abundances of bacterial genera throughout the fermentation process. Synergistic effects were showed by the Phytolin+SCFiber and Polynol+SCFiber combinations on specific bacterial genera, which included the increases of beneficial bacteria and inhibitions of potential pathogenic bacteria. While SCFiber was effective in increasing total SCFA produced during fermentation, its combination with sugarcane polyphenols from Phytolin and Polynol affected the production dependent on the phenolic contents. These changes in SCFA production were postulated to be associated with specific metabolic pathways that are predicted to be possessed by different abundant bacteria present within the gut microbiota community. I further investigate the effects of the major flavones in sugarcane, luteolin, tricin, diosmetin and diosmin, to define the polyphenol-fiber interactions more clearly, particularly in providing firm evidence of synergistic action on the gut microbiota between the two components using the in vitro pig fecal fermentation system. In their respective combinations with SCFiber, these flavones caused unique gut microbiota profile by changing the operational taxonomic units counts and alpha- and beta-diversities and leading to synergistic modulatory effects on the relative abundances of specific bacterial genera throughout the in vitro fermentation. In addition, associations of different bacterial taxa were found within different treatments as compared to the baseline bacterial community. Depending on the flavone, their combinations with SCFiber also synergistically improve the productions of specific SCFA. Overall, this thesis described my research in elucidating the potential of using SCFiber as a natural carrier for the delivery of bioactive sugarcane polyphenols to the colon, with a view to improving colon health by the modulation of the gut microbiota towards a better profile and increasing SCFA production. It also provides the fundamental perspectives to the study of potential synergistic interaction between fiber and polyphenols with the gut microbiota.
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    Phenotypic investigation of Saccharomyces cerevisiae morphogenesis and the effects of sourdough yeast-bacteria interactions on colony morphology
    Winters, Michela Pia ( 2022)
    The yeast Saccharomyces cerevisiae is a prominent model organism and instrumental in several industrial and food fermentations. The morphological state of the yeast has significant impacts on how it operates within these applications. Thus, greater understanding of how S. cerevisiae regulates its morphology is critical in maintaining and improving the yeasts functions. Particularly, within the field of sourdough fermentation, understanding how S. cerevisiae behaviour is affected by the presence of different microbial species allows better prediction of the effects of this mixed culture on bread quality. Therefore, this thesis aims to investigate the control of morphogenesis in S. cerevisiae and how colony morphology is impacted by sourdough yeast-bacteria interactions. The morphological switch to filamentous growth in S. cerevisiae is claimed to occur through the intercellular signalling mechanism of quorum sensing. However, the vague definitions surrounding quorum sensing makes the presence of this mechanism uncertain in the yeast. In this thesis, I begin by proposing more precise definitions for intercellular signalling and quorum sensing. I, then, use these criteria to critically analyse prior research in the area. A lack of evidence was found to support a critical signal concentration triggering morphogenesis and failure to use physiological concentrations of putative signal molecules. A novel methodology to address these research gaps was developed, which used polarised budding as a proxy for filamentous growth. This allowed the identification of the critical cell density and physiological metabolite concentration present when cells switched to more polarized growth. Results indicated that only non-physiological concentrations of the putative quorum sensing molecule, 2-phenylethanol, induced this morphological switch. Therefore, a quorum sensing mechanism was not supported, and a toxicity mechanism to induce polarised budding was proposed. Sourdough fermentation to produce bread is still a relatively new research area. To date, no studies have characterised the morphological phenotypes of sourdough S. cerevisiae isolates. Therefore, I investigated a range of phenotypes in previously uncharacterised sourdough isolates and compared them to laboratory isolates. Established assays were applied to investigate the phenotypes of colony and mat morphology, mat formation, agar invasion, pseudohyphal growth, polarized budding and metabolite production. The lack of correlation found between the phenotypes highlights their matrix- and strain-dependency. Differences in metabolite production between sourdough and laboratory yeast isolates suggests that sourdough isolates are more likely to produce better quality bread. Sourdough interkingdom interactions were investigated by analysing the effect of co-culturing sourdough S. cerevisiae and bacteria on colony morphology. The presence of bacteria caused growth inhibition and filamentous growth in the yeast. Limosilactobacillus fermentum also underwent morphogenesis when grown in the presence of yeast. While preliminary, these results reveal novel effects of yeast-bacteria interactions that should be explored in further studies. Overall, the results presented here further fundamental knowledge of the control of morphogenesis in laboratory and sourdough strains of S. cerevisiae. This can be applied to understand similar processes in higher organisms and is relevant to improving fermented food and beverage quality and bio-ethanol production.
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    Understanding Protists and their Relationships with Antibiotic Resistance in Soil Ecosystems
    Nguyen, Thi Bao Anh ( 2022)
    The rapid spread of antibiotic resistance genes (ARGs) in the environment is a major global concern to public health. Soils are one of the largest reservoirs of ARGs. Although antibiotic resistance is considered as a natural mechanism of microorganisms before synthetic antibiotic usages, there is little knowledge about biological factors, especially predation, as a potential driver of antibiotic resistance in natural habitats. The majority of previous studies have exclusively focused on the relationships between bacteria, fungi, human and abiotic factors with the antibiotic resistance, but overlooked soil protists, a pivotal component of soil food webs and primary microbial predators of bacteria and fungi. Soil protists play important roles in regulating microbial structure, nutrient cycling and plant performance. Protists can influence community structure and activities of their microbial prey (i.e., bacteria and fungi) while being subjected to predation of invertebrates. Nevertheless, the contribution of biotic and abiotic factors as well as the relative importance of trophic regulations (including top-down and bottom-up controls) in shaping the community structure and functional traits of protists in natural soil ecosystems remain unclear. Additionally, secondary metabolites, e.g., antibiotics, are considered to be excreted by bacteria to fight against the predation of protists. However, how antibiotics influence soil protists and their functional groups as well as roles of soil protists in the resistance of bacteria in natural settings are surprisingly unknown. Therefore, the research project applied the advanced amplicon sequencing and high throughput quantitative PCR (HT-qPCR) to assess drivers of the protist diversity and community structure across natural soil ecosystems, as well as the relationships of soil protists with bacterial antibiotic resistance, antibiotics and/or antibiotic-amended livestock manure in agricultural soil systems. In the first large-scale study, we assessed impacts of abiotic (climatic and edaphic properties) and biotic (vegetation types, bacteria, fungi and invertebrates) factors on the core protist community using DNA sequencing on 258 soil samples across eastern and northern Australian natural soils. Core protist community, including abundant and ubiquitous taxa, were constituted by consumers and phototrophs as major functional groups. The supergroups Alveolata, Rhizaria and Archaeplastida dominated the core protist community. The results revealed that invertebrate and bacterial communities were identified as top predictors for the diversity and community composition of core consumers, whereas mean annual temperature best explained the diversity and community composition of core phototrophs. In the second large-scale study, we further identified novel findings about the relative contribution of trophic regulations on the structure of the whole protist communities. The best polynomial fit model revealed that bacterial and invertebrate diversity were important drivers of the alpha diversity of functional groups of protists. Moreover, the compositions of protistan taxonomic and functional groups were better predicted by bacteria and fungi, than by soil invertebrates. There were strong trophic interconnections between protists and bacteria in multiple organismic network analysis. In general, the study provided new evidence that bottom-up control of bacteria played an important role in shaping the soil protist community structure, which can be derived from feeding preferences of protists on microbial prey, and their intimate relationships in soil functioning or environmental adaptation. In a microcosm incubation, we estimated effects of two widely used antibiotics, oxytetracycline and ciprofloxacin, on the protistan and bacterial communities in an arable soil. Rhizaria were identified as the most abundant supergroup of soil protists, followed by Amoebozoa, Stramenopiles, and Aveolata. Consumers were the predominant functional group of the protistan community. Soil protists were more resistant to antibiotics than bacteria with an insignificant change in the protist diversity, contrasting to the decreasing bacterial diversity upon antibiotic exposures. However, the antibiotics significantly reduced the relative abundance of the dominant supergroups of protistan consumers Rhizaria and Amoebozoa, while the relative abundance of other consumer and phototrophic protists were increased. In brief, this work provided novel experimental evidence that the bacterivorous consumers, an important functional group of protists, were more sensitive to antibiotics than other functional groups. Furthermore, through a 130-day microcosm incubation with cattle and poultry manures amended with or without an antibiotic tylosin, we recorded strong responses of soil protists to manure application with a significant reduction in their alpha diversity in all treatments. Significant temporal changes were found in the alpha diversity and composition of soil protists and their functional groups. Stronger effects on protists were detected in soil samples incubated with tylosin-amended manure. Moreover, many consumer, phototrophic and parasitic taxa were highly sensitive to all manure treatments at days 50 and 130. Altogether, these incubation studies demonstrated the negative effects of the common antibiotics and livestock manures on soil protists. To unravel protists’ effects on the profile of antibiotic resistance genes (ARGs) and bacterial community, we established a soil microcosm incubation by inoculating three different protist concentrations (low, medium and high) in soil microcosms within 90 days in the final incubation study. Our results indicated that the higher abundance and number of ARGs were found in high protist treatments, compared to low and medium treatments. High protist concentrations significantly enriched the abundance and number of dominant ARGs encoding main mechanisms of antibiotic resistance, including efflux pump and antibiotic deactivation. Notably, the abundance of ARGs encoding multidrug (oprJ and ttgB genes) and tetracycline (tetV) efflux pump was enhanced by 608.39%, 723.99% and 3,051.78%, respectively, in high treatments, which suggests strong effects of soil protists on promoting the antibiotic resistance. We identified that the potential protist predation-driven increase in bacterial antibiotic resistance was further paralleled by enhanced abundance of numerous bacterial genera under the high pressure of protists. Our novel findings demonstrated that soil protists are an important factor promoting the development of the antibiotic resistance of bacterial community, which advanced our understanding of the biological driving forces for the evolution and development of antibiotic resistance. In conclusion, through the high-throughput sequencing and quantitative PCR, this research project provided wide-spectrum evidence and systematic understanding of the diversity and community structure of topsoil protists across natural and agricultural systems. The large-scale studies demonstrated the importance of biotic factors, typically bottom-up control of bacteria, in structuring the core and whole communities of protist and their functional traits in natural soil systems, with potential implications in structuring the community of other soil organisms in terrestrial ecosystems. These studies also indicated strong trophic relationships between protists, bacteria and other soil microbes, which suggest that the antibiotic resistance and soil functions need to be interpreted by different trophic groups (especially bacteria and protists) across the soil food web. Moreover, the sensitivity of soil protists to antibiotics and/or antibiotic-amended animal manure, as well as the enrichments of soil resistome and numerous bacterial taxa in three soil incubation studies demonstrated that soil protists are a crucial driver of the bacterial antibiotic resistance in soil ecosystems, implying their potential effects in regulating other bacterial activities. This research project sheds light on the importance of predators as a biological driving force of the antibiotic resistance and implications for their regulation in other soil functions and ecological processes in natural settings. Altogether, this work advances our knowledge about roles of the biological interactions in shaping the bacterial antibiotic resistance in nature, as well as facilitates the mitigation and management of potential environmental and health issues caused by the antibiotic resistance in future.
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    Exploring Microwave Heat Treatment Mechanisms And Effect On Forage Hay Feeding Value, Digestion, Nitrogen Partitioning And Weight Gain Of Sheep
    Shishir, Md Safiqur Rahaman ( 2022)
    The main objective of this PhD study was to investigate the microwave (MW) treatment effect on different forage hays' nutritive value and its subsequent feeding effect on sheep performance. Pepsin-cellulase in vitro digestibility was conducted to explore the effects of MW heat treatment on different types of forage hays’ nutritive value (Chapter 3). Apparent in vivo digestibility and nitrogen balance studies were conducted to explore MW-treated forage hay feeding effect on sheep intake, digestibility, nitrogen utilisation and predicted live weight gain (Chapter 4). In order to understand the potential mechanisms underpin observed changes in hay nutritive value and sheep performance, the scanning electron microscopic (SEM) image (Chapters 3 and 4) and dielectric properties of hay were analysed (Chapter 5). The in vitro study dry matter (DM) % of forage hays increased with increasing MW treatment time (P < 0.001). The improved in vitro DM digestibility and digestible organic matter in the DM was only recorded from MW-treated lucerne (60 s), wheat (40 s), and canola (20 s) (P < 0.001), with no changes observed in pasture and oat hay. A major observation of this study was a positive relationship between the MW energy required for maximal DM digestibility improvement and the baseline crude protein content of forage hay (R2 = 0.79; P < 0.001). The sheep performance study showed that DM intake and predicted live weight gain was higher in MW treated lucerne hay group than control (P < 0.001). The digestibility of DM and OM both increased by 8% in MW-treated lucerne hay compared to the control (P <0.001). The digestibility of neutral detergent fibre, acid detergent fibre (ADF) and nitrogen increased by 11%, 9% and 10%, respectively, in MW-treated lucerne hay, compared to the control (P < 0.001). The MW treatment increased nitrogen retention (P = 0.037) and microbial nitrogen synthesis (P = 0.047) in the lucerne hay group more than in control. A limited effect of MW treatment on the wheat hay-fed sheep group was observed. Forage hays SEM images showed clear cell microstructure destruction (P <0.001) due to MW treatment in lucerne hay but not in wheat hay. The dielectric measurement showed that with increasing moisture content, both the dielectric constant and the dielectric loss factor increased for roughages and concentrates feed. However, the responses were not linear. The oven-dried plant samples’ dielectric properties were very low compared with those of the higher moisture content samples. Thus, the sample’s moisture content was the dominant contributor to the feed samples’ dielectric behaviour, which links to the efficiency of utilising MW power to alter hay quality. Overall, the PhD studies showed that MW heat treatment could be applied as a novel processing method to improve some forage hays’ digestibility and subsequent sheep performance. Such beneficial changes occurred might be due to variations in dielectric properties of hays and cell microstructure destruction, which not only improve forage hays’ nutritive value but also allows better nutrient accessibility by rumen microbes. However, future study is required to predict the optimum MW treatment energy power/time needed for improving forage hay nutritive value and animal performance.
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    Healthy Chocolates Enriched with Microencapsulated Probiotics
    HOSSAIN, MD NUR ( 2022)
    Probiotics are live microorganisms that can modulate the gastrointestinal environment and confer health benefits to the host when ingested in sufficient numbers. However, maintaining probiotic viability with the minimum recommended number during processing, storage, and exposure to the harsh gastrointestinal tract is very challenging in food and pharmaceutical product development. Encapsulation has been considered an effective technique to improve probiotic viability, and various polymers including, alginate, casein, pectin, chitosan, xanthan, and gelatin have been used as encapsulants. In this study, the probiotics were encapsulated using a complex admixture of cocoa mass along with Sodium alginate and fructooligosaccharides to extend the probiotic’s storage viability and survivability during the gastrointestinal transit. Cocoa powder or chocolates is a complex mixture of polysaccharides, lipids, polyphenols, and other organic compounds that are not readily digestible and absorbed in the gastrointestinal tract. The aim of this PhD work was to investigate cocoa powder as an encapsulating ingredient to overcome the major challenges of processing and storage conditions and adverse gastrointestinal conditions. Furthermore, the project was involved with the formulation of healthy probiotic chocolates which can modulate the gut environment to produce some essential metabolites. Two different types of chocolates (70 and 45 percent cocoa chocolates) and eight different probiotics named Lactobacillus casei 431, Lactobacillus rhamnosus, Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668, Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Streptococcus thermophilus UASt 09, and Lactobacillus delbrueckii subsp. bulgaricus 12315 were used in the entire study. The results revealed excellent improvement in the survivability of probiotics and demonstrated their role in modulating the gut environment and the production of short chain fatty acids and vitamin B12 and the bioconversion of polyphenols during the in vitro gastrointestinal digestion and colonic fermentation. The coca powder admixture was used to encapsulate probiotics and compared with commonly used encapsulating materials such as whey protein concentrate, hi maize resistant starch, and skim milk powder with an emulsion based freeze drying technique. The encapsulation efficiency of the cocoa powder along with sodium alginate and fructooligosaccharides showed very promising results as compared with whey protein concentrate, hi maize resistant starch, skim milk, and sodium alginate. Encapsulating Lactobacillus rhamnosus, Lactobacillus casei 431, Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668, and Bifidobacterium animalis subsp. lactis using cocoa powder along with sodium alginate and fructooligosaccharides demonstrated a very good yield and a high rate of survivability. The best viabilities of encapsulated probiotics in chocolates during storage were found to be 120 days at 4 degrees celsius and 90 days at room temperature. Additionally, a high survivability rate of the cocoa powder encapsulated probiotics was detected at 60 degrees celsius which could be excellent findings to scale up the production of probiotic chocolates industrially. The in vitro gastrointestinal digestion and colonic fermentation of two types of probiotic chocolates with 70 and 45 percent cocoa chocolates contents showed positive effects on the gut environment modulating activity. All tested probiotic chocolates demonstrated very promising results concerning the bioaccessible polyphenols and bioconversion activity, production of fecal metabolites such as short-chain fatty acids (acetic, propionic, isobutyric, butyric, and isovaleric acid), and biosynthesis of vitamin B12 (Lactobacillus sanfranciscensis and Lactobacillus plantarum). However, the interaction among the various tested probiotics and chocolate polyphenols (45 and 70 percent cocoa) enriched with these encapsulated probiotics during the in vitro gastrointestinal digestion and colonic fermentation revealed different levels of bioconversion of polyphenols (bioaccessible polyphenols). For example, Lactobacillus sanfranciscensis JCM5668 and Steptococcus thermophilus UASt 09 demonstrated better conversion activities for epicatechin, procyanidin B1, and procyanidin B2 in probiotic chocolate containing 70 perccent cocoa, while Lactobacillus casei 431, Lactobacillus rhamnosus, Lactobacillus plantarum UALp 05 showed better capacities with 45 percent cocoa chocolates. These findings concluded that chocolates could be utilized by probiotics for their metabolic activities and modulate the gut environment which improved the functionality of chocolates. Furthermore, investigating the impact of in vitro gastrointestinal digestion and colonic fermentation of probiotic chocolate on the production of short chain fatty acids revealed a direct relationship between the probiotics and the released short chain fatty acids. The acetic acid production rate was much higher for Lactobacillus plantarum UALp 05 and Bifidobacterium animalis subsp. lactis than other probiotics at 24 to 48 hours. While Lactobacillus sanfranciscensis JCM5668 and Streptococcus thermophilus UASt 09 produced fewer amounts of acetic acids. Similarly, different amounts of propionic acid were produced by different probiotics after 48 hours of colonic fermentation and ranked from largest to smallest as follows: Bifidobacterium animalis> Lactobacillus acidophilus> Streptococcus thermophilus> Lactobacillus sanfranciscensis> Lactobacillus bulgaricus> Lactobacillus casei> Lactobacillus rhamnosus> Lactobacillus plantarum irrespective of cocoa content. The best isobutyric acid efficacy was estimated in probiotic-chocolate fortified with Streptococcus thermophilus followed by Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei containing 45 percent cocoa chocolates at 24 hours of colonic fermentation. Lactobacillus acidophilus was the most efficient producer of butyric acid and released significantly higher amounts than all other probiotics throughout the colonic fermentation. However, Lactobacillus acidophilus produced a smaller amount of both isobutyric and isovaleric acids. Surprisingly, 70 percent of dark chocolate samples produced a higher amount of branched short chain fatty such as isobutyric and isovaleric acid. These secreted short chain fatty acids during the in vitro gastrointestinal digestion and colonic fermentation of probiotic chocolates may indicate that chocolate could act as prebiotics for the gut microbiota, which then produced more short chain fatty acids. Vitamin B12 was the other assessed metabolite. All eight tested probiotics with both types of chocolates (45 and 70 percent cocoa) were investigated for the biosynthesis of vitamin B12. Results showed that in vitro colonic fermentation produced vitamin B12 only in the presence of Lactobacillus plantarum UALp 05, Lactobacillus sanfranciscensis JCM5668. Additionally, these probiotics produced more B12 in the presence of 45 percent cocoa chocolate than 70 percent dark chocolate. The sensory tasting using a 9 point hedonic scale and five sensory attributes (taste, color, flavor, texture, and overall acceptability) of the formulated probiotic chocolates containing 45 percent and 70 percent cocoa powder showed insignificant differences between these two types of probiotic chocolates. Consequently, it was concluded that both probiotic chocolate with 45 percent and 70 percent cocoa contents could be attractive to consumers. Such probiotic chocolate will offer consumers a new source of healthy snacks with additional health benefits. This thesis presents promising research on probiotics with chocolate as a carrier and expands the research window for probiotic functional products using non dairy chocolates as carriers. It is recommended that conducting a follow up study on probiotic chocolate at an industrial scale with in vivo applications will provide stronger proof of the functional properties and health benefits of such a healthy chocolate snack.
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    Integrated assessment of global and regional Nr mitigation: potential, cost-benefits, and strategies
    Zhang, Xiuming ( 2022)
    Nitrogen (N) is an important component in plant and animal growth, Human activity has dramatically altered the global N cycle, resulting in excessive reactive nitrogen (Nr) lost to the environment, which has far exceeded the safe boundary of the earth, caused serious environmental problems, threatening human survival. Agriculture is considered a major source contributor of excess Nr emissions and therefore this sector should be highly looked to for solutions. However, the previous environmental policies mainly focus on NOx emissions and mitigation, which is mainly related to fossil fuel burning and industrial production. In contrast, ammonia (NH3) emissions and mitigation were less noticed, and there are limited NH3 policies and incentives in place globally. Although recent research has paid attention to reducing agricultural Nr emissions, they mainly focus on certain Nr emission types with certain mitigation measures. An integrated and comprehensive assessment of Nr mitigation (mainly NH3) is lacking in most regions of the world. In this context, this thesis performs a comprehensive assessment of the global and regional agricultural Nr mitigation with a focus on the NH3 mitigation and croplands via a multi-model coupling framework. Findings include: (1) An uncertainty assessment of the N budget: a case study of China To address issues with food security and environmental protection, an accurate N budget is a prerequisite for efficient N management and fact-based decision-making. However, significant disparities exist among previous estimates of N budgets and fluxes flow. Taking China, the world’s largest Nr emitter as an example, this thesis attempts to improve the understanding of uncertainties in N budgets, including N inputs and outputs, and N loss from diffident systems from 1980 to 2018 via the CHANS N balance model. Results show varying uncertainties in major N fluxes across spatial scales. The activity levels of agriculture and industry and their corresponding Nr emission factors are the most sensitive factors for the total N budget uncertainty in China. A proposed framework that combines bottom-up N balance estimates and top-down measurements and validation models could well constrain the uncertainties of N budgeting. (2) A first comprehensive assessment of NH3 mitigation in China China contributes to one-third of global anthropogenic ammonia (NH3) emissions due to its intensive agriculture. The engagement of NH3 mitigation is required for further advancement of air quality and public health in China. However, China has not yet developed and implemented regulations to minimize NH3 emissions, and there is no comprehensive evaluation of NH3 mitigation available. Based on the improved accuracy of the N budget of China, this study performed the first comprehensive assessment of national NH3 mitigation. According to the findings, China's agricultural NH3 emissions have a technological mitigation potential of 53% (38-67%), which is about twice as high as Europe's (24%). and the societal benefits of halving agricultural NH3 emissions in China far exceed the abatement costs. In addition to technical mitigation options, reducing the consumption of animal products could provide additional NH3 mitigation potential. (3) A first comprehension assessment of NH3 mitigation in Australia Australia is one of the world's leading producers and exporters of agricultural products. 40-60% of N inputs into the Australian agricultural system are lost to the environment. NH3 is the primary form of gaseous N loss, making it significant to explore the necessity and feasibility of NH3 mitigation for policy reference. This study maps the most recent spatial-temporal NH3 emissions in Australia and evaluates the viability of NH3 emission reductions. The findings indicate that the implementation cost to achieve 32% of Australia's potential abatement is $3.1 (0.5-7.6) billion, or 10% of the overall abatement benefit. The ecosystem benefits of reducing NH3 emissions outweigh the benefits for human health because of the distinctiveness and significance of its biodiversity. Animal and farming systems should be given priority in Australia's NH3 emission reduction efforts to take advantage of low-hanging fruit. (4) An integrated assessment of mitigating N pollution from global croplands with cost-effective measures Mitigating N pollution on croplands globally is a very difficult task due to the nature of nonpoint source pollution coming from millions of farms and the difficulties in putting Nr reduction measures into practice. This study included 1521 field observations from throughout the world and found 11 important strategies that might increase crop yield and NUE by 10–30% and 10-80%, respectively, while decreasing on-farm N losses by 30–70%. Overall, smart adoption of these options worldwide might result in a 20% boost in crop yields and a 32% decrease in N pollution to the environment. Given the disparity between costs and benefits to the entire society compared to farmers, it may be required to develop innovative policies, such as a nitrogen credit system (NCS), to choose, encourage, and, if necessary, subsidize farms’ implementation of these options. In conclusion, this thesis provided a comprehensive assessment of agricultural Nr (mostly NH3) mitigation on a regional and worldwide scale. The viability and necessity of implementing cost-effective interventions are justified by the strong mitigation benefit-to-cost ratios. The results of this thesis help to bridge the gap between research, policymaking, and agricultural practices and encourage cross-disciplinary discussion about reducing Nr emissions.
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    Surface Modification of Coal and its Application to Mitigate Ammonia Loss from Livestock Manure
    Zhang, Wei ( 2022)
    Nearly all global ammonia (NH3) emissions are emitted from agricultural sources, including ammonia-based fertilizers and livestock manure, which can have significant negative impacts on both human health and the natural environment. Ammonia emissions from livestock manure represent a large loss of nitrogen (N) nutrients that would otherwise be available for plant growth. Application of lignite has been demonstrated to be a practical and cost-effective method to reduce NH3 emissions from cattle feedlots. However, widescale implementation of such technology is limited because lignite mines and intensive livestock production systems are not always located near each other and the high water content of lignite makes the long distance transport uneconomical. Therefore, this thesis investigates the feasibility of using a thermal air oxidation method to dewater lignite and to surface modify the more commonly occurring and geologically abundant black coal (BC) and coal tailings (CTs) from coal washing process as alternative materials to lignite to reduce NH3 loss from livestock manure. The first phase of the research determined the optimum oxidation temperature for lignite dewatering and BC surface modification and evaluated the gaseous NH3 and aqueous ammonium (NH4+) adsorption before and after treatment. Lignite treated at 200 degrees Celsius exhibited the highest adsorption capacities of 76.4 and 3.7 mg g-1 for NH3 and NH4+, respectively. The water content of lignite was decreased from 61.6 to 4.2% with an enhanced apparent activation energy of combustion, suggesting that the thermal oxidation process would not increase the spontaneous combustion risk of lignite. Characterization of the surface chemistry indicated that these enhancements originated from the partial oxidation of lignite surfaces. The acidic surface functional groups on lignite played an important role in NH4+ adsorption. Similarly, the thermal air oxidation method used in lignite dewatering was then employed for the surface modification of a BC. The NH3 adsorption capacity of BC treated at 300 degrees Celsius achieved an 11-fold increase (49.7 mg g-1) compared with the untreated BC. The concentration of acidic surface functional groups on BC was significantly increased after treatment. Moreover, NH3 adsorption capacity showed a linear relationship with the concentration of acidic surface functional groups, indicating NH3 adsorption of BC was enhanced due to interactions between NH3 and acidic functional groups from thermal air oxidation. In the second phase, the dewatered lignite and surface-modified BC (treated at 200 and 300 degrees Celsius, respectively) displayed the greatest NH3 adsorption capacity were chosen to investigate the capacity of these materials (applied at 30%) to reduce NH3 loss from livestock manure through a laboratory incubation experiment. Results showed that dewatered lignite and surface-modified BC reduced NH3 volatilization from cattle manure to a similar extent as the raw lignite. Moreover, the adsorption and immobilization of manure ammoniacal N induced by coal materials were identified as key drivers in reducing NH3 loss from manure, outweighing the pH effect. In the phase three, CTs were treated at different temperatures and varying duration to investigate the reaction kinetics of formation of acidic surface functional groups on coal surfaces and elucidate the NH3 adsorption mechanisms. The CT treated at 300 degrees Celsius for 5 hours showed an NH3 uptake of up to 52.5 mg g-1, which was a 25-fold increase in comparison to the untreated CT. Spectroscopic analysis showed that the acidic surface functional groups such as carboxylic groups present on treated CT surfaces could react with NH3 via an acid-base reaction leading to the formation of NH4+ but they were also involved in the formation of amides. A relatively low activation energy of 50.2 kJ mol-1 for the formation of acidic surface functional groups on CTs was obtained using an Arrhenius analysis in the temperature range of 200 – 300 degrees Celsius of oxidation, indicating that thermal air oxidation is a simple, rapid, and effective surface modification method to generate acidic surface functional groups on coal surfaces to capture NH3. Overall, the findings of this study provide a fundamental insight into the effectively design and development of coal-derived adsorbent materials to capture NH3 and show promise for future utilization of modified coal materials for mitigation of manure NH3 emissions in livestock farms.
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    Positive Human-Animal Interactions, Early Life Experiences and Stress Resilience in Pigs
    Lucas, Megan Elinor ( 2022)
    Pigs in intensive production systems routinely encounter several challenging situations including abrupt weaning, painful husbandry procedures, intense contact with stockpeople, and exposure to novel social and physical environments. The ability of pigs to cope with these routine stressors has implications for their welfare and productivity and may be affected by their previous experiences with humans. Furthermore, experiences that occur early in life, including interactions with the dam, the physical environment as well as early experiences with humans, may have a profound effect on the development of stress coping mechanisms that impacts how pigs cope with stress throughout their lives. The aim of this thesis was to examine the effects of positive interactions with humans and early life human and housing experiences on stress resilience in pigs. Stress resilience was measured based on: 1. behavioural and physiological responses to stressors such as routine husbandry and management practices, isolation, novelty, and humans; 2. basal behavioural and physiological measurements that reflect how pigs cope with their general environment; and 3. measurements of biological fitness. Overall, positive interactions with humans, including patting, stroking and scratching pigs, reduced pigs’ fear of humans and their stress responses to routine husbandry and management practices imposed by stockpeople. Sows that received 2 min of daily positive human contact during gestation showed less avoidance of and more interaction with stockpeople imposing pregnancy testing and vaccination in the home pens, and piglets that received 3 or 5 minutes of regular positive human contact showed less escape behaviour and vocalisations during husbandry procedures in the lactation period. Providing opportunities for positive human interaction from 0-4 wk of age was extremely effective in reducing fear of humans, based on positively handled piglets showing increased approach and interaction with an unfamiliar human at 2 and 3 wk of age. Although the effects of handling on fear of humans appeared to weaken over time, there was evidence of early positive human contact resulting in pigs showing less fearful responses to a human at 6, 9 and 14 wk of age, indicating that some reduction in fear of humans lasted well beyond the period when the handling treatment was imposed. In addition to reducing fear of humans, there was evidence that early positive human contact conferred broader effects on stress resilience. Early positive handling reduced pigs’ fear of a novel object at 3 wk of age, escape behaviour after weaning, and cortisol concentrations after weaning and after isolation at 7 wk of age. Furthermore, early positive handling reduced pigs’ injuries and increased serum concentrations of brain-derived neurotrophic factor (BDNF) at 4 wk of age, with limited evidence indicating similar effects on these measurements much later in life at 17 wk of age. The early housing environment had a considerable effect on the human-animal relationship, with pigs reared in farrowing crates showing less fear of humans at 2, 3, 4 and 6 wk of age compared to pigs reared in loose farrowing and lactation pens with more space, physical complexity, and opportunity for interaction with the sow. During the lactation period, piglets in loose pens showed more play behaviour and less repetitive nosing of pen mates and had higher concentrations of serum BDNF compared to piglets in farrowing crates, but surprisingly, they were less able to cope with several stressors before and after weaning. During the lactation period, loose pen piglets showed more avoidance of a novel object, a greater intensity of escape behaviour during capture by a stockperson, and more injuries at 2 wk of age. After weaning, loose pen pigs showed more escape behaviour, less inactivity and had higher cortisol concentrations. At 7 wk of age, loose pen pigs showed more vocalising in a novel area and had higher cortisol concentrations after isolation, and at 21 wk of age, they showed more baulking when being moved out of the home pen by a stockperson. This research showed that positive interactions with humans can ameliorate the stress associated with routine husbandry practices involving stockpeople. When imposed early in life, positive handling had an extended effect on reducing pigs’ fear of humans and appeared to foster stress resilience in a general capacity, based on fewer injuries, higher BDNF concentrations and reduced responses to challenges such as novelty, weaning and isolation in positively handled pigs. In addition to reducing fear of humans through habituation and conditioning, brief and regular close human contact early in life may provide a minor challenge to overcome, that improves the competence of pigs to cope with other types of challenges faced in the future. This research also demonstrated considerable differences in the stress resilience of pigs reared in the two housing treatments studied. While piglets in the loose system showed more play behaviour and had higher BDNF concentrations during the lactation period (both of which are linked to stress resilience) they showed far less flexibility in response to stressors such as routine husbandry practices, weaning, isolation, and exposure to novelty and humans. Piglets in the loose system were reared in a more isolated environment with less contact with people and other pigs and less visual stimulation in general. This may have resulted in loose pen piglets having fewer opportunities to learn to cope with small challenges such as frequent and/or close exposure to stockpeople and other pigs, thus increasing their vulnerability to stress. In addition, heightened maternal responses of loose pen sows towards stockpeople may have increased loose pen piglets’ fear of humans. More research is needed to determine the specific features of the early housing environment that affect both immediate and long-term fear and stress responses of pigs. Overall, this research: 1. showed that the early human and housing environment for pigs can have both immediate and longer-term consequences on stress resilience; and 2. contributed to a growing body of work showing that humans are a key determining factor in the welfare of pigs.
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    Effect of Heat Stress on Sheep Meat Quality
    Zhang, Minghao ( 2022)
    Heat stress (HS) is one of the greatest challenges facing the global livestock industry. An increase in global temperature and relative humidity (RH) is likely to compromise animal welfare, production, and meat quality during hot summer months. While the negative effects of chronic HS on sheep are well established, the influence of short-term HS are comparatively less known and especially, there are few studies reporting impact of HS on sheep meat quality which is still not clear. Therefore, this PhD project was designed to demonstrate the effect of various heat exposure durations on meat quality of sheep and identify the differences, if any, among different sheep breeds. This PhD thesis consists of three animal experiments designed to investigate the effect of short-term HS on physiology and meat quality of different sheep breeds. Because the duration of heat exposure which could lead to negative impact of HS on meat quality of different sheep breeds was not clear, the first animal study was a preliminary experiment to confirm the negative effects of HS period on different sheep breeds meat quality. In this experiment, two wool type (Merino, Southdown) and two hair type (Dorper and Wiltshire) breeds of sheep (n=5 of each breed) were allocated to either thermoneutral [TN; 18-21 C, 45-55% relative humidity (RH)], or heat stress (HS; 28 C-38 C, 40-60% RH) conditions (n=10 of each group) for two weeks to assess the effect of two weeks HS on physiological, blood, and meat quality parameters. Compared with TN, two weeks HS significantly increased the respiration rate (RR) and rectal temperature (RT) of sheep but had no impact on retail meat colour, water holding capacity (WHC) and Warner-bratzler shear force (WBSF) for five days high oxygen map packaging display. Among four breeds, Merino showed higher L*, b* and purge loss than other breeds, Merino and Dorper had higher cooking loss than Southdown and Wiltshire. This study provided some preliminary evidence that HS can cause negative effects on meat quality of sheep and needs to be investigated further to select an adaptive and productive sheep breed for changing climate. The second animal experiment compared the heat tolerance capacity of 2nd cross and Dorper lambs after two weeks HS treatment based on growth, physiological and retail meat quality parameters. Forty-eight Dorper and 2nd cross [Poll Dorset x (Border Leicester x Merino)] lambs (38-42 kg; 4-5 months old) were allocated to either TN (18-21 C, 45-55% RH), or HS (28 C–38 C, 40-60% RH) conditions in a 2 x 2 factorial design for two weeks. Compared with 2nd cross, Dorper lambs had a lower (RR) and rectal temperature (RT) and exhibited less decline in body weight under HS. 2nd cross lambs showed a higher body weight gain than Dorper under TN conditions. HS increased a* and chroma of the Longissimus thoracis et lumborum (LTL) of second cross lambs over 10 days retail display but had no impact on Dorper lambs. In conclusion, Dorpers showed higher heat tolerance compared with second cross lambs during the two weeks HS exposure condition. The third animal experiment assessed the influence of short duration heatwaves (HW) on sheep physiological, blood biochemical profile and retail meat quality parameters. Seventy-two second-cross lambs (Poll Dorset x (Border Leicester x Merino)) were selected and exposed to either one, three or five days of stimulation heatwaves (HW) or TN (28-38 C and 40-60% RH; 18-21 C, 40-55% RH) conditions in climate-controlled chambers. Lambs exposed to one to five days of HW exhibited higher RR, RT, skin temperatures (ST) and heart rates (HR) compared to lambs exposed to an equal duration of TN conditions. HW also had significant impact on blood gas parameters which included higher blood pH, and lower CO2, Ca2+, Na+, HCO3- and Cl-. However, lambs’ ileal histology and GSSG:GSH of intestine and muscle was not influenced by HW. Longissimus thoracis (LT) muscle showed higher total antioxidant capacity (TAC) in HW condition, but the TAC of intestine and blood had no difference between HW and TN groups. In terms of determinants of meat quality, HWs had no significant effects on muscle metabolism (rate and extent of pH decline, muscle glycogen and lactate content) and meat quality (cooking loss and shear force). Similarly, there were limited impacts of one to five days of HW on the colour (L*, a*, b* and R630/580) and drip loss of the LTL and semimembranosus (SM) muscles during four days’ overwrap retail display. These results suggest that short-duration HWs (one to five days) lead to significant negative effects on sheep physiology but may not effect postmortem muscle metabolism and meat quality. In conclusion, this thesis demonstrated that short-term HS (less than two weeks) are a significant challenge for sheep physiology and welfare but have limited impact on some of the meat quality attributes while have no impact on other meat quality parameters (water holding capacity, shear force etc). These results are explained by the fact that sheep can cope with short duration by recruiting some physiological and behavioral responses which are not severe enough to cause oxidative damage to muscle or intestinal tissues which are seen under chronic heat stress. Compared with wool type breeds, hair type breeds have better heat tolerance and are less affected by short-term heat stress.